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The current study was carried out to evaluate the effect of motor activity (hypokinesia) limitation on the body and organ systems. Hypokinesia contributes to changes in the work of many adaptive systems of the body, which leads to disorders in the musculoskeletal system, respiratory, muscular, and cardiovascular systems. Of particular relevance and practical interest is the effect on the body not only of hypokinesia, but also the processes during the transition from it to an active motor regime (recovery period). The study was carried out on 2 groups of white outbred rats: group 1 - animals growing under limited motor activity for 30 days, group 2 - animals in the recovery period after 30 days of hypokinesia. The study of the functional parameters of the Langendorff heart of rats (LVP, HR, CF) was carried out on the selected groups. Results of the study revealed that after 30 days of hypokinesia and 14 days recovery period, the heart rate decreased 1.2 times and the coronary flow decreased 2 times. Further, the current study has shown a tendency to restore the heart rate parameters of an isolated heart but the restoration of the coronary flow and cardiac contractility has not been identified. The result of the study suggested that 14 days is not a full-fledged period for the restoration of the functional parameters of the body. This study devoted the effect of motor activity (hypokinesia) limitation on the body and organ systems are relevant and significant.

Extrahepatic tissues which oxidise ketone bodies also have the capacity to accumulate them under particular conditions. We hypothesised that acetyl-coenzyme A (acetyl-CoA) accumulation and altered redox status during low-flow ischaemia would support ketone body production in the heart. Combining a Langendorff heart model of low-flow ischaemia/reperfusion with liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), we show that β-hydroxybutyrate (β-OHB) accumulated in the ischaemic heart to 23.9 nmol/gww and was secreted into the coronary effluent. Sodium oxamate, a lactate dehydrogenase (LDH) inhibitor, increased ischaemic β-OHB levels 5.3-fold and slowed contractile recovery. Inhibition of β-hydroxy-β-methylglutaryl (HMG)-CoA synthase (HMGCS2) with hymeglusin lowered ischaemic β-OHB accumulation by 40%, despite increased flux through succinyl-CoA-3-oxaloacid CoA transferase (SCOT), resulting in greater contractile recovery. Hymeglusin also protected cardiac mitochondrial respiratory capacity during ischaemia/reperfusion. In conclusion, net ketone generation occurs in the heart under conditions of low-flow ischaemia. The process is driven by flux through both HMGCS2 and SCOT, and impacts on cardiac functional recovery from ischaemia/reperfusion.

The estimation of wound age and wound vitality is a recurring task in forensic routine work and has been subject of forensic research for a long time. By now, an unrestrictedly reliable marker or set of markers has not been found. In a study on myocardial infarctions, matrix metalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) were detected immunohistochemically in mechanically wounded myocardium (ECG electrodes, vessel ligations). Against this background, the potency of MMP-9, MMP-2, and TIMP-1 as markers for the estimation of wound age and wound vitality was tested in a broad approach with human tissue samples drawn during autopsies and with an animal model, the isolated perfused Langendorff heart. The study comprised samples of injured human skeletal muscle, injured human myocardium, rats’ hearts with vital wounds, and rats’ hearts with postmortem-inflicted wounds that were all stained immunohistochemically. The results showed great scattering, leading to the conclusion that MMP-2, MMP-9, and TIMP-1 are not suitable for wound age estimation. Merely the results for TIMP-1 suggested that this marker might be able to differentiate between vital and postmortem-inflicted wounds. With a view to the promising results of the preceding study, the results underline the necessity to test possible markers of wound age/wound vitality on a large and diverse sample set.

Recent studies revealed that relatively small changes in perfusate sodium ([Na+](o)) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+](o) modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+](o) that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia-reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia-reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+](o) decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+](o) at baseline and reduced the time to peak ischemic contracture. Ischemia-reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+](o) further depressed mechanical function. In summary, elevating [Na+](o) by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+](o), with cardiac ischemia may require further investigation. National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01-HL141855, R01-HL138003, R01-HL102298, F31-HL147438, R01-HL132236, R01-HL123647] This study was supported by National Institutes of Health F31-HL147438 awarded to DRK, National Institutes of Health R01-HL132236 awarded to JWS, National Institutes of Health R01-HL123647 awarded to DAB, and National Institutes of Health R01-HL141855, R01-HL138003, R01-HL102298 awarded to SP.

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The custom-designed intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 × 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dual-wavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state.NEW & NOTEWORTHY Here, a novel nondestructive real-time approach for monitoring intrinsic indicators of cardiac metabolism and oxygenation is described using a catheter-based transillumination of the left ventricular free wall together with complete spectral analysis of transmitted light. This approach is a significant improvement in the quality of cardiac optical absorbance spectroscopic metabolic analyses.

Graphical abstract, Oscar Langendorff introduced the first method for isolating a heart with contractile activity in 1895. Since then, the Langendorff method has remained a powerful technique in cardiac research and has led to major advances in medicine. The primary goal of the Langendorff method is to provide an isolated heart with oxygen and metabolites via a cannula inserted into the aorta. The Langendorff heart is a complex in vitro technique used primarily in pharmacological and physiological research that allows the evaluation of multiple cardiac hemodynamic parameters including, but not limited to, contractility and heart rate. This article will first provide a brief background of the Langendorff method as well as details regarding organ isolation. Finally, the article will discuss the benefits of a new technique for hanging the isolated heart aorta and the benefits of this technique over traditional methods

The present study aimed to investigate the effects of remifentanil during adaptation followinsg myocardial ischemia, and its possible clinical applications. Remifentanil was used during the simulation of adaptation following ischemia, which was performed using a Langendorff heart perfusion system. A total of 75 rats were divided into five groups, and the coronary flow, cardiac output and the cardiac enzyme content in coronary effluent prior to ischemia and post-reperfusion were recorded. Electron microscopy was used to observe myocardial ultrastructure, and the volume of aortic and coronary effluent was also measured. The recovery of cardiac output upon reperfusion was significantly higher following remifentanil treatment (91%), as compared with the ischemic control group (78%; P

The aim of this study was to prove whether albumin-derived perfluorocarbon-based nanoparticles (capsules) can operate as a novel artificial oxygen carrier in a rat Langendorff-heart perfusion model. Hearts perfused with capsules showed increased left ventricular pressure and rate pressure product compared to hearts perfused with pure Krebs-Henseleit (KH)-buffer. The capsules prevented the myocardium from functional fail when in their absence a noxious ischemia was observed. Capsules did not change rheological properties of KH-buffer and could repeatedly reload with oxygen. This albumin-derived perfluorocarbon-based artificial oxygen carrier preserved the function of rat hearts due to the transport of oxygen in a satisfactory manner. Because of these positive results, the functionality of the applied capsules should be verified in living animals.

Aim. To study the cardioprotective effects of exenatide and vildagliptin on the doxorubicin model of cardiomyopathy. Material and methods. In the experiments at the isolated the Langendorff heart of the rat, the cardioprotective effect of exenatide (10 μg/kg/day) (Baeta®, Eli Lilly and Company, USA) and vildagliptin (0.2 mg/kg/day) (Galvus®, Novartis, Switzerland), on the contractile function of an isolated heart that underwent anterior doxorubicin (20 mg/kg, intraperitoneally for 48 hours) pathology was evaluated. Cardioprotective effect was assessed by the results of a functional test with highfrequency stimulation (480 bpm) under conditions of hypercalcium (5 mmol) perfusion. Results. The results show that the exenatide (10 μg/kg/day) and vildagliptin (0.2 mg/kg/day) incretin mimetics show a cardioprotective effect on the doxyrubicin pathology model, which is expressed in a decrease in the coefficient of diastolic dysfunction (StTTI), respectively, to 5.3±0.1 cu and 6.5±0.2 cu compared with the control group 8.3±0.1 cu. Conclusion. An assumption is made about the way of realization of the cardioprotective effect of incretin mimetics by increasing the expression of gem-oxygenase-1 (HO-1). This prevents the heme-catalyzed formation of highly active hydroxyl radicals from hydrogen peroxide. Induction of heme oxygenase-1 is accompanied by an increase in ferritin activity, which has an antiapoptotic effect due to the fact that ferritin binds excess free iron in cells subjected to oxidative stress.

We validated a motion field imaging (MFI) assay with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) as a model to assess multiple cardiac liabilities by comparing the guinea-pig Langendorff heart with hiPS-CMs using 4 reference compounds and 9 internal compounds. We investigated repolarization duration, beating rate (BR), conduction speed, contractility, and inhibitory profile of three cardiac ion channels: hERG, Cav1.2, and Nav1.5. For repolarization, the contraction-relaxation duration (CRDc) of hiPS-CMs was generally consistent with the QTc interval of Langendorff heart. However, 2 internal compounds shortened CRDc despite QTc prolongation in Langendorff heart. Cardiac ion channel profiling revealed that hiPS-CMs could not be used to detect QTc prolongation when the value of Cav1.2 IC50 / hERG IC50 for a compound was between 1 and 10, whereas hiPS-CMs showed responses largely consistent with Langendorff heart when Cav1.2 IC50 / hERG IC50 was below 1 or above 10. The accuracy of hiPS-CMs for the BR was not high, mainly because the BR of hiPS-CMs was increased by an inhibition of Cav1.2. The hiPS-CMs were highly sensitive to conduction speed and contractility, able to detect QRS widening caused by Nav1.5-inhibition, as well as decreased LVdP/dtmax caused by the inhibition of Cav1.2 and/or Nav1.5. In conclusion, the MFI assay with hiPS-CMs would be useful for evaluating multiple cardiac liabilities. The ion channel profile helps to interpret the results of MFI assay and correctly evaluate cardiac risks. Therefore, an integrated cardiac safety assessment with MFI and ion channel profiling is recommended.

Introduction Cardiovascular diseases are the most frequent cause of death in the western world. During myocardial infarction both, ischemia and reperfusion after therapeutic intervention cause myocardial damages, resulting in ischemia/reperfusion-injury (I/R-injury). Research therefore searches cardio protective interventions, aiming to reduce myocardial injury and following consequences such as heart failure. A promising method, named Remote Ischemic Preconditioning (RIPC), consists of transient local ischemia distant from the heart. Its cardio-protective effects were already proved in basic research but failed in clinical trials. Purpose The aim of the study is to examine the impact of cardiac risk factors like diabetes mellitus (DM) or age on effectiveness of RIPC in order to identify distinct groups of patients who might benefit from RIPC in the clinical setting and to reveal the effectiveness of cardioprotection delivered by plasma samples of these groups of patients. Methods RIPC (3 x 5 min arm ischemia, induced by an inflated blood pressure cuff) was performed on young and healthy volunteers and elderly patients with coronary heart disease with or without diabetes mellitus. Blood was taken before (Baseline) and 30 minutes after the intervention. The coronary system of murine hearts in a Langendorff heart model was perfused using dialysate from the human plasma obtained before and after RIPC. This was followed by 40 minutes of global ischemia. After 60 minutes of reperfusion left ventricular function was analysed. The infarct size was measured after 120 minutes reperfusion time using a TTC staining. Results Addition of dialysate of plasma obtained from healthy volunteers after RIPC to the coronary system of Langendorff perfused murine hearts significantly improved the recovery of left ventricular developed pressure (39±15.1 vs 26±8.5% of baseline levels, p=0.04) and reduced the infarct size (37.5±15.6 vs 21.2±11.4% of the ventricular volume, p=0.03) compared to baseline-dialysate treated controls. While plasma dialysate obtained from elderly patients with DM undergoing RIPC failed to show significant cardio protective effects of RIPC, murine hearts treated with plasma dialysate of elderly patients with coronary heart disease without diabetes after undergoing RIPC showed significant improvement of cardiac function (20±6.5 vs 24±7% of baseline levels, p=0.02) and infarct size (33.2±8.0 vs 11.1±2.5% of the ventricular volume, p=0.01). Conclusion The cardio-protective effect of RIPC on myocardial function and infarct size after I/R is influenced by diabetes but not by age or the presence of coronary heart disease. In consequence, RIPC might protect patients with coronary heart disease without DM but not diabetic patients from cardiac I/R injury. This important finding potentially explains why RIPC yielded promising results in various basic science experiments but failed to show cardio protective effects in clinical trials. Acknowledgement/Funding Forschungskommission of the Faculty of Medicine of the Heinrich-Heine-University Düsseldorf

Cocaine-related deaths occur regularly in forensic routine work. In cases in which the detected concentration of cocaine is rather low and other causes of death apart from intoxication can be ruled out, the question arises if adulterants of cocaine might have played a crucial role. In the present study, cardiac effects of cocaine, of the adulterant levamisole and of mixtures of both were evaluated using the isolated perfused Langendorff heart. While exposed to the substances, functional parameters heart rate, left ventricular pressure and coronary flow were documented. Relevant alterations of these parameters were found for cocaine as well as for levamisole. Exposing the hearts to a mixture of both resulted in a combination of these effects; the emergence of new alterations or an obvious aggravation were not detected. Nevertheless, the results imply that the consumption of cocaine adulterated with levamisole bares an increased risk for cardiac complications, especially in the presence of preexisting cardiac pathologies.

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS–based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

Arterial perivascular adipose tissue (PVAT) can elicit vasodilator signals complementary to those elicited by the endothelium in SHRSP.Z- Leprfa/IzmDmcr (SHRSP.ZF) rats, an animal model of metabolic syndrome (MetS). Here, we tested whether a glucose cotransporter 2 inhibitor (SGLT2-i; tofogliflozin) increased this PVAT effect to prevent the deterioration of cardiac function in aging SHRSP.ZF rats. Tofogliflozin treatments (1 or 10 mg/kg/day) or vehicle (control) were administered for 10 weeks by oral gavage to SHRSP.ZF rats, starting at 13 weeks of age. At 23 weeks of age, glucose levels in the serum and urine (24 h after the last administration) were determined using commercial kits. Vasodilator responsiveness of PVAT-surrounded or PVAT-free superior mesenteric arteries was determined using acetylcholine with organ-bath methods. Cardiac ventricular function and coronary flow were determined using Langendorff heart preparations. Serum and urine glucose levels in SGLT2-i treatment groups did not differ from those in the controls, but the ratios of glycated to non-glycated albumin were lower than those in the controls. Tofogliflozin treatments did not alter relaxations in the presence of PVAT or affect relaxations of PVAT-free arteries. Left ventricular systolic pressures, maximum rate of pressure decline, and coronary flow in ex vivo hearts did not differ among the treatment groups. PVAT effects and cardiac dysfunction were not altered by tofogliflozin treatment in SHRSP.ZF rats with MetS. These results do not provide strong evidence to support the use of SGLT2-i as a cardiovascular protective therapy in MetS, which occurs prior to the onset of type 2 diabetes.

Gαi-coupled receptors play important roles in protecting the heart from ischemic injury. Regulator of G protein signaling (RGS) proteins suppress Gαi signaling by accelerating the GTPase activity of Gαi subunits. However, the roles of individual RGS proteins in modulating ischemic injury are unknown. In this study, we investigated the effect of RGS6 deletion on myocardial sensitivity to ischemic injury. Hearts from RGS6 knockout (RGS6−/−) and RGS6 wild-type (RGS6+/+) mice were subjected to 30 minutes of ischemia and 2 hours of reperfusion on a Langendorff heart apparatus. Infarcts in RGS6−/− hearts were significantly larger than infarcts in RGS6+/+ hearts. RGS6−/− hearts also exhibited increased phosphorylation of β2-adrenergic receptors and G protein–coupled receptor kinase 2 (GRK2). Mitochondrial GRK2 as well as caspase-3 cleavage were increased significantly in RGS6−/− hearts compared with RGS6+/+ hearts after ischemia. Chronic propranolol treatment of mice prevented the observed increases in ischemic injury and the GRK2 phosphorylation observed in RGS6−/− hearts. Our findings suggest that loss of RGS6 predisposes the ventricle to prodeath signaling through a β2AR-GRK2–dependent signaling mechanism, and they provide evidence for a protective role of RGS6 in the ischemic heart. Individuals expressing genetic polymorphisms that suppress the activity of RGS6 may be at increased risk of cardiac ischemic injury. Furthermore, the development of agents that increase RGS6 expression or activity might provide a novel strategy for the treatment of ischemic heart disease.

Aim We studied whether available oxygen without induced mechanical stretch regulates the release of the biologically active B-type natriuretic peptide (BNP) from Langendorff heart. Methods Rat hearts were isolated and perfused with a physiological Krebs–Henseleit solution at a constant hydrostatic pressure in Langendorff set-up. The basal O2 level of perfusate (24.4 ± 0.04 mg L−1) was gradually lowered to 3.0 ± 0.01 mg L−1 over 20 min using N2 gas (n = 7). BNP and O2 level were measured from coronary flow. During control perfusions (n = 5), the O2 concentration was kept at 26.6 ± 0.3 mg L−1. Results A low oxygen concentration in the perfusate was associated with a significant increase in BNP release (F = 40.4, P

INTRODUCTION The purpose of this study was to examine the effects of rapamycin on the cardioprotective effect of hypoxic preconditioning (HPC) and on the mammalian target of rapamycin (mTOR)-mediated hypoxia-inducible factor 1 (HIF-1) signaling pathway. MATERIAL AND METHODS Primary cardiomyocytes were isolated from rat pups and underwent rapamycin and/or HPC, followed by hypoxia/re-oxygenation (H/R) injury. Cell viability and cell injury were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and qRT-PCR was used to measure HIF-1α and mTOR mRNA expression. A Langendorff heart perfusion model was conducted to observe the effect of rapamycin. RESULTS Rapamycin treatment nearly abolished the cardioprotective effect of HPC in cardiomyocytes, reduced cell viability (p = 0.007) and increased cell damage (p = 0.032). HIF-1α and mTOR mRNA expression increased in cardiomyocytes undergoing I/R injury within 2 h after HPC. After rapamycin treatment, mTOR mRNA expression and HPC-induced HIF-1α mRNA expression were both reduced (p < 0.001). A Langendorff heart perfusion model in rat hearts showed that rapamycin greatly attenuated the cardioprotective effect of HPC in terms of heart rate, LVDP, and dp/dtmax (all, p < 0.029). CONCLUSIONS Rapamycin, through inhibition of mTOR, reduces the elevated HIF-1α expression at an early stage of HPC, and attenuates the early cardioprotective effect of HPC.

Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H2O2-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1–8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30–1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury. Keywords: Ischemia/reperfusion injury, Langendorff heart, Reactive oxygen species, Tumstatin

Zebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research., Summary: Here, we describe a new experimental tool to assess contractility of the isolated zebrafish heart perfused in a Langendorff-like system, which will facilitate using zebrafish as a model for human cardiomyopathies.

Background and purpose In drug research using the rat Langendorff heart preparation, it is possible to study left ventricular (LV) contractility using an intraventricular balloon (IVB), and arrhythmogenesis during coronary ligation-induced regional ischaemia. Assessing both concurrently would halve animal requirements. We aimed to test the validity of this approach. Experimental approach The electrocardiogram (ECG) and LV function (IVB) were recorded during regional ischaemia of different extents in a randomized and blinded study. Key results IVB-induced proarrhythmia was anticipated, but in hearts with an ischaemic zone (IZ) made deliberately small, an inflated IVB reduced ischaemia-induced ventricular fibrillation (VF) incidence as a trend. Repeating studies in hearts with large IZs revealed the effect to be significant. There were no changes in QT interval or other variables that might explain the effect. Insertion of an IVB that was minimally inflated had no effect on any variable compared with ‘no IVB’ controls. The antiarrhythmic effect of verapamil (a positive control drug) was unaffected by IVB inflation. Removal of an inflated (but not a non-inflated) IVB caused a release of lactate commensurate with reperfusion of an endocardial/subendocardial layer of IVB-induced ischaemia. This was confirmed by intracellular 31phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy. Conclusions and implications IVB inflation does not inhibit VF suppression by a standard drug, but it has profound antiarrhythmic effects of its own, likely to be due to inflation-induced localized ischaemia. This means rhythm and contractility cannot be assessed concurrently by this approach, with implications for drug discovery and safety assessment.

Kaempferol exerts cardioprotective actions through incompletely understood mechanisms. This study investigated the molecular mechanisms underlying the cardioprotective effects of kaempferol in sinus node dysfunction (SND) heart. Here, we demonstrate that angiotensin II (Ang II) infusion causes SND through oxidized calmodulin kinase II (CaMKII). In contrast to this, kaempferol protects sinus node against Ang II-induced SND. Ang II evoked apoptosis with caspase-3 activation in sinus nodal cells. However, kaempferol lowered the CaMKII oxidization and the sinus nodal cell death. To block the CaMKII oxidization, gene of p47phox, a cytosolic subunit of NADPH oxidase, was deleted using Cas9 KO plasmid. In the absence of p47phox, sinus nodal cells were highly resistance to Ang II-induced apoptosis, suggesting that oxidized-CaMKII contributed to sinus nodal cell death. In Langendorff heart from Ang II infused mice, kaempferol preserved normal impulse formation at right atrium. These data suggested that kaempferol protects sinus node via inhibition of CaMKII oxidization and may be useful for preventing SND in high risk patients.

OBJECTIVES: The loss of effectiveness of ischaemic preconditioning in protecting old hearts from ischaemia/reperfusion damage is thought to be due to low sirtuin 1 levels in old hearts. We sought to determine whether resveratrol (RES), an activator of sirtuin 1, would restore this protection to that seen with ischaemic preconditioning in young hearts. METHODS: A Langendorff heart perfusion model was established in 80 old and 80 adult rats to test the effects of hypoxic preconditioning (HPC) and/or RES on preventing hypoxia–reperfusion (H/R) injury. The effects were further tested by comparing the effects of HPC and RES on cell survival rate and lactate dehydrogenase (LDH) in cardiomyocytes from 15 old and 15 young humans. RESULTS: The HPC + RES group performed better in both adult and old groups than the corresponding H/R, HPC and RES groups, causing 50% in the adult and 40% in the old group restoration of left ventricular developed pressure and 90% in the adult and 80% in the old group restoration of dp/dtmax. HPC and RES each reduced apoptosis in both groups. The HPC + RES treatment showed an additive benefit in reducing apoptosis in the adult group but not in the old group. In H/R-treated young and old human cardiomyocytes, cell survival and LDH level were significantly improved in the RES + HPC group compared with the HPC group. CONCLUSIONS: This study showed that RES lessened the ageing effect and enhanced the cardioprotective effect of HPC in older individuals.

Matrixmetalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of metalloproteinases (TIMP) 1 were tested as markers of myocardial early ischemia/infarctions. Experiments with an animal model, the isolated Langendorff heart, and analysis of human tissue samples drawn during autopsies were performed. Results of the experiments with the Langendorff model implied that the detectable amount of the markers might increase early after the onset of ischemia, in less than one hour, under ideal conditions. The results of the examined human cases showed that MMP-2 is constantly detectable in human myocardial tissue with an increased amount in case of an infarction with longer survival times. MMP-9 and TIMP-1 were negative in control cases, distinct positive staining results were obtained mainly in cases of infarctions with longer survival times and only rarely in those with a short survival time. According to these results MMPs and TIMPs do not qualify as first choice markers of myocardial infarctions. As an interesting side finding in the Langendorff experiments, positive staining results for all three markers were seen in myocardial areas that were mechanically traumatized by ECG-electrodes or ligation of blood vessels. These findings make the markers interesting for forensic wound age estimation.

Diabetes mellitus (DM) is associated with an electrical remodeling of the heart, increasing the risk of arrhythmias. However, knowledge of electrical remodeling in the sinoatrial node (SAN) by DM is limited. We investigated the expression of HCN channel isoforms, HCN1–HCN4, in SAN from streptozotocin (STZ)-induced diabetic rats and the age-matched controls. We found that the STZ-induced diabetic rats have a lower intrinsic heart rate, a lengthened sinoatrial conduction time, and rate-corrected maximal sinoatrial node recovery time in vivo as well as a longer cycle length (CL) in vitro, as compared with the control. Optical mapping of the SAN demonstrated an inferior leading pacemaker site, reduced SAN conduction velocity and diastolic depolarization slope, and a longer action potential duration in the STZ-induced diabetic rats than in the control. The transcripts and proteins of HCN2 and HCN4 in diabetic SAN were reduced. Specific blockade of HCN channels by 3 μmol/L ivabradine significantly prolonged the CL of a Langendorff heart by 18% in the diabetic rats and 26% in the control. The reduced expression of HCN channel isoforms in the SAN of the STZ-induced diabetic rat may be an important contributor to the reduced SAN function in DM.

New Findings •What is the topic of this review? The topic of the review is the intrinsic cardiac nervous system in the rabbit. •What advances does it highlight? The anatomy of rabbit intrinsic ganglia is similar to that of other species, including humans. Immunohistochemistry confirms the presence of cholinergic and adrenergic neurones, with a striking arrangement of neuronal nitric oxide synthase-positive cell bodies. Activation of atrial ganglia produces effects on local and remote regions. Heart disease is a primary cause of mortality in the developed world, and it is well recognized that neural mechanisms play an important role in many cardiac pathologies. The role of extrinsic autonomic nerves has traditionally attracted the most attention. However, there is a rich intrinsic innervation of the heart, including numerous cardiac ganglia (ganglionic plexuses), that has the potential to affect cardiac function independently as well as to influence the actions of the extrinsic nerves. To investigate this, an isolated, perfused, innervated rabbit Langendorff heart preparation was considered the best option. Although ganglionic plexuses have been well described for several species, there was no full description of the anatomy and histochemistry of rabbit hearts. To this end, rabbit intrinsic ganglia were located using acetylcholinesterase histology (n = 33 hearts). This revealed six generalized ganglionic regions, defined as a left neuronal complex above the left pulmonary vein, a right neuronal complex around the base of right cranial vein, three scattered in the dorsal right atrium and a region containing numerous ventricular ganglia located on the conus arteriosus. Using immunohistochemistry, neurons were found to contain choline acetyltransferase or tyrosine hydroxylase and/or neuronal nitric oxide synthase in differing amounts (choline acetyltransferase > tyrosine hydroxylase > neuronal nitric oxide synthase). The function of rabbit intrinsic ganglia was investigated using a bolus application of nicotine or electrical stimulation at each of the above sites whilst measuring heart rate and atrioventricular conduction. Nicotine applied to different ganglionic plexuses caused a bradycardia, a tachycardia or a mixture of the two, with the right atrial plexus producing the largest chronotropic responses. Electrical stimulation at these sites induced only a bradycardia. Atrioventricular conduction was modestly changed by nicotine, the main response being a prolongation. Electrical stimulation produced significant prolongation of atrioventricular conduction, particularly when the right neuronal complex was stimulated. These studies show that the intrinsic plexuses of the heart are important and could be crucial for understanding impairments of cardiac function. Additionally, they provide a strong basis from which to progress using the isolated, innervated rabbit heart preparation.

Since information regarding the effects of pH on the extent of nifekalant-induced repolarization delay and torsades de pointes remains limited, we assessed it with a Langendorff heart model of guinea pigs. First, we investigated the effects of pH change from 7.4 to 6.4 on the bipolar electrogram simulating surface lead II ECG, monophasic action potential (MAP), effective refractory period (ERP), and terminal repolarization period (TRP) and found that acidic condition transiently enhanced the ventricular repolarization. Next, we investigated the effects of pH change from 6.4 to 7.4 in the presence of nifekalant (10 μM) on the ECG, MAP, ERP, TRP, and short-term variability (STV) of MAP90 and found that the normalization of pH prolonged the MAP90 and ERP while the TRP remained unchanged, suggesting the increase in electrical vulnerability of the ventricle. Meanwhile, the STV of MAP90 was the largest at pH 6.4 in the presence of nifekalant, indicating the increase in temporal dispersion of repolarization, which gradually decreased with the return of pH to 7.4. Thus, a recovery period from acidosis might be more dangerous than during the acidosis, because electrical vulnerability may significantly increase for this period while temporal dispersion of repolarization remained increased. Keywords:: nifekalant, acidosis, Langendorff, torsades de pointes, monophasic action potential

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [14C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 expression was most abundant in heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

β-adrenoceptor antagonists are commonly used in ischaemic heart disease (IHD) patients, yet may impair signalling and efficacy of 'cardioprotective' interventions. We assessed effects of chronic β1-adrenoceptor antagonism on myocardial resistance to ischaemia-reperfusion (IR) injury and the ability of cardioprotective interventions [classic ischaemic preconditioning (IPC); novel sustained ligand-activated preconditioning (SLP)] to reduce IR injury in murine hearts. Young male C57Bl/6 mice were untreated or received atenolol (0.5g/l in drinking water) for 4 weeks. Subsequently, two cardioprotective stimuli were evaluated: morphine pellets implanted (to induce SLP, controls received placebo) 5 days prior to Langendorff heart perfusion, and IPC in perfused hearts (3×1.5min ischaemia/2min reperfusion). Atenolol significantly reduced in vivo heart rate. Untreated control hearts exhibited substantial left ventricular dysfunction (~50% pressure development recovery, ~20mmHg diastolic pressure rise) with significant release of lactate dehydrogenase (LDH, tissue injury indicator) after 25min ischaemia/45min reperfusion. Contractile dysfunction and elevated LDH were reduced >50% with IPC and SLP. While atenolol treatment did not modify baseline contractile function, post-ischaemic function was significantly depressed compared to untreated hearts. Atenolol pre-treatment abolished beneficial effects of IPC, whereas SLP protection was preserved. These data indicate that chronic β1-adrenoceptor blockade can exert negative effects on functional IR tolerance and negate conventional IPC (implicating β1-adrenoceptors in IR injury and IPC signalling). However, novel morphine-induced SLP is resistant to inhibition by β1-adrenoceptor antagonism.

The isolated retrograde-perfused Langendorff heart and the isolated ejecting heart have, over many decades, resulted in fundamental discoveries that form the underpinnings of our current understanding of the biology and physiology of the heart. These two experimental methodologies have proven invaluable in studying pharmacological effects on myocardial function, metabolism, and vascular reactivity and in the investigation of clinically relevant disease states such as ischemia-reperfusion injury, diabetes, obesity, and heart failure. With the advent of the genomics era, the isolated mouse heart preparation has gained prominence as an ex vivo research tool for investigators studying the impact of gene modification in the intact heart. This review summarizes the historical development of the isolated heart and provides a practical guide for the establishment of the Langendorff and ejecting heart preparations with a particular emphasis on the murine heart. In addition, current applications and novel methods of recording cardiovascular parameters in the isolated heart preparation will be discussed. With continued advances in methodological recordings, the isolated mouse heart preparation will remain physiologically relevant for the foreseeable future, serving as an integral bridge between in vitro assays and in vivo approaches.

The purpose of this investigation was to determine the effects of acute physiological GSH administration on endothelium-mediated reduction in coronary vascular resistance (CVR) using isolated perfused Sprague-Dawley rat hearts. A dose-response curve to GSH was conducted to determine a threshold concentration of GSH. We demonstrate that 30 μM GSH was sufficient to reduce CVR, and maximal dilation was achieved with 1 mM. In subsequent experiments, GSH was administered at concentrations of 0 [control (CON)], 1 μM, or 10 μM (GSH10), and dose-response curves to the endothelial agonist bradykinin (BK) were constructed. These GSH concentrations were chosen because of the physiological relevance and because the effects of GSH on BK action could be assessed independent of baseline differences in CVR. Sensitivity to BK (EC50) was enhanced in GSH10 vs. CON ( P < 0.05). This enhancement remained in the presence of nitric oxide (NO) synthase inhibition l-ωnitro-l-arginine (lNAME) and/or soluble guanylate cyclase (sGC) inhibition. Treatment with 4-hydroxy (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPOL) enhanced the sensitivity to BK in CON, similar to the effects of GSH10 and GSH10 + TEMPOL. However, the GSH10-dependent enhancement of EC50 observed in the presence of lNAME did not occur in the presence of lNAME + TEMPOL or in the presence of lNAME + sGC inhibition and NO scavenging. Collectively, these results suggest that GSH enhances BK-mediated dilation and reduction in CVR through an antioxidant-dependent mechanism that involves a NO intermediate but is unrelated to acute production of NO and GC-dependent effects of NO. These results suggest a mechanism whereby physiologically relevant levels of GSH modulate the endogenous reactive oxygen species and NO control of endothelium-dependent coronary vascular function.

BACKGROUND AND PURPOSE Rosiglitazone is an anti-diabetic drug improving insulin sensitivity and glucose uptake in skeletal muscle and adipose tissues. However, several recent clinical trials suggest that rosiglitazone can increase the risk of cardiovascular ischaemia, although other studies failed to show such risks. Therefore, the effects of rosiglitazone on the coronary circulation and any potential vascular targets need to be elucidated. Here, we show that the vascular isoform of the ATP-sensitive K+ (KATP) channel is inhibited by rosiglitazone, impairing physiological regulation of the coronary circulation. EXPERIMENTAL APPROACH The KIR6.1/SUR2B channel was expressed in HEK293 cells and studied in whole-cell and inside-out patch configurations. The Langendorff heart preparation was used to evaluate rosiglitazone in the coronary circulation of wild-type (WT) and KIR6.1-null (Kcnj8−/−) mice. KEY RESULTS KIR6.1/SUR2B channels in HEK cells were inhibited by rosiglitazone in a membrane-delimited manner. This effect was markedly enhanced by sub-micromolar concentrations of glibenclamide and the IC50 for rosiglitazone fell to 2µM, a therapeutically achievable concentration. In the Langendorff heart preparation rosiglitazone inhibited, concentration-dependently, the coronary vasodilation induced by isoprenaline, without affecting basal coronary tone. Effects of rosiglitazone on coronary perfusion were attenuated by more than 50% in the Kcnj8−/− mice, supporting the involvement of KATP channels in this effect of rosiglitazone on the coronary circulation. CONCLUSIONS AND IMPLICATIONS These results indicate that the vascular KATP channel is one of the targets of rosiglitazone action, through which this drug may compromise coronary responses to circulating vasodilators and perhaps also to metabolic stress.

Problem statement: Palmitate is a known cardiac lipotoxin that blunts cardiomyocyte contractile function and induces apoptosis, likely via accumulation of the lipotoxic ceramide. Ceramide is a sphingolipid and localizes to caveolae, which are lined in the inner membrane leaflet by caveolin proteins. In this study, we investigated the effects of palmitate on caveolin proteins and on endothelial Nitric Oxide Synthase (eNOS), a signaling mediator that binds to caveolin-3, the musclespecific caveolae scaffolding protein. Approach and Results: Mice fed a high palmitate diet for 12 weeks showed pathologically increased coronary flow in the ex vivo Langendorff heart especially at low extracellular calcium concentrations. In these hearts, eNOS Ser1177 phosphorylation was increased compared to standard or high fat control diet hearts. This suggested that eNOS, a potent vasodilator in the heart, is affected by palmitate. In vitro experiments showed that exposure of HL-1 cardiomyocytes to palmitate causes translocation of eNOS from the plasma membrane to a per-inuclear location and causes an 80% decrease in Thr495 phosphorylation. This corresponded with a 41% decrease in NO production. To determine the mechanism of the loss of plasma membrane bound eNOS, we investigated the effect of palmitate on caveolin-3 and found decreased caveolin-3 protein levels by 70% compared to control cells. The remaining 30% of caveolin-3 was localized to a peri-nuclear location. In contrast to previous studies, palmitate did not cause apoptosis in cardiomyocytes. Conclusion: Overall, we show for the first time that a high palmitate diet leads to loss of caveolin-3 in cardiomyocytes and to coronary dysfunction of the mouse heart, via uncoupling of eNOS.

The aim of this study was to clarify whether administration of antipsychotic perphenazine contributes to the electrophysiological changes of the isolated heart. Fourteen adult male Wistar rats were divided into two groups. Langendorff hearts were perfused with Krebs-Henseleit solution at constant pressure (85 mmHg) and 37 °C (CaCl2, 1.2 mM). The electrogram was recorded by touch-free method. The hearts of the first group were exposed to 3.10-5 M perphenazine, the hearts of the second group to 3.10-8 M perphenazine. The incidence of arrhythmias and the heart rate and QT interval changes were studied on electrogram during 30 min periods of control, the first perphenazine administration, washout, and the second drug administration. Perphenazine administration significantly prolonged QT (p < 0.001) and QTc (p < 0.05) in group 1. In group 2, QT and QTc prolongation was less pronounced (p < 0.05). A number of arrhythmias appeared, from single premature ventricular complexes to more severe ones in both groups. The heart rate changes were non-significant. We conclude that although phenothiazines are still medicaments of choice in certain psychoses, their cardiovascular side effects should be always taken in consideration.

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease.

Introduction Cardiac liabilities represent a major cause of recent withdrawal of marketed drugs and also for the high attrition rate evidenced during late stage drug development. To identify molecules with potential cardiovascular risks early in drug development, a screening model of ex vivo Langendorff hearts has been validated with 26 reference compounds of various chemical and therapeutic classes. Methods The hearts of adult guinea pigs were maintained by retrograde perfusion in Langendorff mode, beating spontaneously at sinus rhythm or paced via the right atrium at 200 and 300 beats per minute. Multiple parameters consisting of hemodynamic function (coronary and left ventricle pressure), cardiac electrophysiology (electrocardiogram and monophasic action potential) and indices of arrhythmogenesis (triangulation, reverse-use dependence, repolarization dispersion and beat-to-beat instability), together with overt arrhythmia were evaluated simultaneously. Ascending concentrations up to either 100-fold of the determined hERG IC50 or nominally 100 µM were routinely tested utilizing 4–6 hearts per compound. Results Each compound exhibited a unique cardiovascular profile: (i) the majority displayed concentration and heart rate-dependent mixed-ion channel or multiple-target effects that frequently resulted in bradycardia, atrioventricular block, negative inotropy, coronary vasodilatation, and QRS widening. (ii) Compounds associated with high arrhythmogenic risk in the clinic exhibited more “positive signals” at concentrations within 30-fold of their maximal therapeutic free plasma concentration than those with less arrhythmogenic potential. (iii) For several potent torsadogens, proarrhythmic indices other than the prolongation of QT/QTc and MAP duration appeared more sensitive in representing proarrhythmic liability. (iv) A scoring system was developed to assist in the rank-ordering of potential cardiotoxicants. Discussion The cardiovascular action of reference compounds profiled by this isolated heart model was generally consistent with their known mechanisms and, except for the sinus heart rate, correlated well with that observed in the clinic. Further, the overall cardiac liability estimated by the scoring system matched the clinical documentation, suggesting this model could serve as a valuable tool in early cardiovascular drug safety assessment.

We describe a new approach that combines several techniques to allow abnormal electrical and calcium activity to be visualized within hypoperfused myocardial tissue. A flexible microcannula was inserted into the left anterior descending artery of Langendorff perfused rat hearts, an air-tight seal between the coronary artery and the cannula was created, and an HPLC pump was used to deliver a specified flowrate through the microcannula. High resolution optical mapping of NADH/calcium, NADH/voltage or calcium/voltage was then conducted using a dual camera system. The ECG was acquired using surface electrodes. This perfusion technique is superior to occluding a vessel by either a tie or a clamp because it allows precise control of the composition and amount of flow to a defined ischemic bed. Another advantage is that flow can be stopped and resumed remotely, without touching the heart. This allows ectopic beats, or other arrhythmogenic activity, such as alternans, to be recorded immediately after changes in flow are imposed. Altogether, the described method provides a powerful new tool to assess how coronary flow rate affects the degree of local ischemia by the ability to record abnormal patterns of electrical activity and associated intracellular calcium transients with high spatiotemporal resolution from epicardial areas as small as 100 x 100 microm.